Project Summary Influenza A virus causes disease in 5%-20% of the population with over 200,000 hospitalizations annually in US. The antigen drift and shift of influenza virus due to rapid evolution pose a serious challenge for annual flu vaccination program, which is effective depending on the accurate prediction of the influenza serotypes that will be circulating in the next flu season. The recent failure of influenza vaccine and potential outbreak of influenza pandemics highlights the urgent need for a vaccine that can provide broad protection. Interferon (IFN) is a critical component of the innate immune system and also the bridge between the innate and adaptive immune responses. We recently studied the anti-IFN function of influenza genome using a quantitative and high-throughput genomics system. By incorporating eight IFN-sensitive mutations into influenza genome, we generated a Hyper Interferon Sensitive (HIS) virus as a vaccine candidate. HIS virus is highly attenuated in wild type and immune-deficient SCID mice, but fully competent in IFNAR-deficient mice. HIS provides protection against homologous and heterologous viral challenges. Our central hypothesis is that systematical elimination of IFN-evasion functions on multiple segments of the virus genome generates proper induction of innate immune response, which is essential for establishing long term memory B cell response and T cell response by live attenuated influenza vaccine. Our objective is to determine the minimal replication capacity required for live attenuated influenza virus vaccine, identify and generate single-round infection HIS virus, which can induce strong IFNR signaling in vitro, but has no replication capacity in vivo due to innate immune response. Such vaccine virus candidate would have confined one-round infection during immunization whereas the IFN inducing activity would be strong enough to illicit broad protective immunity. We will generate hyper IFN sensitive virus that has no replication capacity in vivo, and characterize its replication kinetics and responsiveness to IFN in lung epithelial cells. After we obtain such virus, we will infect mice with vaccine candidate viruses and characterize the induced immune responses. Finally, we will determine protection efficacy of vaccine candidate viruses against different strains of influenza virus in vivo. The results achieved from this project will advance our understanding of influenza vaccine development and facilitate the development of universal influenza vaccine.